Absence of long-term balancing selection on variation in EuMYB3, an R2R3-MYB gene responsible for the anther-color polymorphism in Erythronium umbilicatum

Balancing selection has been shown to be common in plants for several different types of traits, such as self-incompatibility and heterostyly. Generally, for these traits balancing selection is generated by interactions among individuals or between individuals and other species (e.g., pathogens or pollinators). However, there are phenotypic polymorphisms in plants that do not obviously involve types of interactions that generate balancing selection. Little is known about the extent to which balancing selection also acts to preserve these polymorphisms. Here we ask whether balancing selection preserves an anther-color polymorphism in Erythronium umbilicatum (Liliaceae). We identified a major gene underlying this polymorphism. We then attempted to detect signatures of balancing selection on that gene by developing a new coalescence test for balancing selection. We found that variation in anther color is in large part caused by variation in a paralog of EuMYB3, an anthocyanin-regulating R2R3-MYB transcription factor. However, we found little evidence for balancing selection having acted historically on EuMYB3. Our results thus suggest that plant polymorphisms, especially those not involved in interactions that are likely to generate negative frequency-dependent selection, may reflect a transient state in which one morph will eventually be fixed by either genetic drift or directional selection. Our results also suggest that regulation of the anthocyanin pathway is more evolutionarily labile than is generally believed.


Table S1
The Erythronium umbilicatum anther-color polymorphism in 16 sites in North Carolina, USA.These 16 sites are distributed along three main rivers/creeks: Eno River, New Hope Creek/Old Field Creek and Morgan Creek.We conducted an anther-color survey during the flowering season in 2017.In each site, we set up 2 m x 2 m plots that were 6 m apart from each other.Within each plot, four 1 m x 1 m quadrats were laid out.We tallied the anther color for all plants in each quadrat, and counted at least 200 plants in each site.The geographical coordinates of each site, the number of flowers surveyed, the number of flowers having purple or yellow anthers, and the percentage of yellow-anthered flowers in each site are shown.

Location
Anther             sample while amplifying EF1α.We obtained the threshold values as the Cq values from the instrument Roche LightCycler 96.The relative expression levels were then calculated as the logarithm of the ratios, Log 10 (R).
Dataset S1 EuMYB3 amino-acid sequences with premature stop codons (indicated by *).The sequence C_P02_1 is from the purple Group 1.2 and is provided as a reference as an exemplar with no premature stop codons.All remaining sequences are from the yellow Groups.Genbank accession numbers for their nucleotide sequences are shown with the sequence names.

Figure S3 (
Figure S3(to be continued on the next page)

Figure S4
Figure S4 The original gel photograph that was cropped to create the panel Fig. 3A "EuEF1α".Dotted rectangle indicates the area shown in Fig. 3A.The100-bp DNA ladder, Plant IDs (referring to Fig.3 for sample information), and a negative control (N) are showed at the top of each lane.The 100-bp, 300-bp and 500-bp fragments of the ladder are labeled.

Figure S5
Figure S5 The original gel photograph that was cropped to create the panel Fig. 3A "EuDfr".Dotted rectangle indicates the area shown in Fig. 3A.The100-bp DNA ladder, Plant IDs (referring to Fig.3 for sample information), and a negative control (N) are showed at the top of each lane.The 100-bp, 300-bp and 500-bp fragments of the ladder are labeled.

Figure S6
Figure S6The original gel photograph that was cropped to create the panel Fig.3A "EuAns".Dotted rectangle indicates the area shown in Fig.3A.The100-bp DNA ladder, Plant IDs (referring to Fig.3for sample information), and a negative control (N) are showed at the top of each lane.The 100-bp, 300-bp and 500-bp fragments of the ladder are labeled.

Figure S7
Figure S7 The original gel photograph that was cropped to create the panel Fig. 3A "EuUf3gt".Dotted rectangle indicates the area shown in Fig. 3A.The100-bp DNA ladder, Plant IDs (referring to Fig.3 for sample information), and a negative control (N) are showed at the top of each lane.The 100-bp, 300-bp and 500-bp fragments of the ladder are labeled.

Figure S8
Figure S8 The original gel photograph that was cropped to create the panel Fig. 3A "EubHLH2".Dotted rectangle indicates the area shown in Fig. 3A.The100-bp DNA ladder, Plant IDs (referring to Fig.3 for sample information), and a negative control (N) are showed at the top of each lane.The 100-bp, 300-bp and 500-bp fragments of the ladder are labeled.The last two lanes are samples for the other experiment.

Figure S9
Figure S9 The original gel photograph that was cropped to create the panel Fig. 3A "EuMYB3".Dotted rectangle indicates the area shown in Fig. 3A.The100-bp DNA ladder, Plant IDs (referring to Fig.3 for sample information), and a negative control (N) are showed at the top of each lane.The 100-bp, 300-bp, 500-bp and 1000-bp fragments of the ladder are labeled.

Figure S10
Figure S10 The original gel photograph that was cropped to create the panel Fig. 3B "EuEF1α".Dotted rectangle indicates the area shown in Fig. 3B.The100-bp DNA ladder, Plant IDs (referring to Fig.3 for sample information), and a negative control (N) are showed at the top of each lane.The 100-bp, 300-bp and 500-bp fragments of the ladder are labeled.

Figure S11
Figure S11 The original gel photograph that was cropped to create the panel Fig. 3B "EuEF1α".Dotted rectangle indicates the area shown in Fig. 3B.The100-bp DNA ladder, Plant IDs (referring to Fig.3 for sample information), and a negative control (N) are showed at the top of each lane.The 100-bp, 300-bp and 500-bp fragments of the ladder are labeled.The last four lanes are samples for the other experiment.

Figure S12
Figure S12The original gel photograph that was cropped to create the panel Fig.3B "EuMYB3".Dotted rectangle indicates the area shown in Fig.3B.The 1-kb DNA ladder, Plant IDs (referring to Fig.3for sample information), and a negative control (N) are showed at the top of each lane.The 0.5-kb, 1-kb, 1.5-kb and 3-kb fragments of the ladder are labeled.

Figure S13
Figure S13 The original gel photograph that was cropped to create the panel Fig. 3B "EuMYB3".Dotted rectangle indicates the area shown in Fig. 3B.The 1-kb DNA ladder, Plant IDs (referring to Fig.3 for sample information), and a negative control (N) are showed at the top of each lane.The 0.5-kb, 1-kb, 1.5-kb and 3-kb fragments of the ladder are labeled.

Table S2
The query sequences used in the BLAST searches.The sequences of Arabidopsis thaliana were retrieved from TAIR (https://www.arabidopsis.org/).Other sequences were retrieved from GenBank.

Table S4
Numbers of gDNA sequences of EuMYB3 in different sequence Groups for purple and yellow individuals.The "Purple" or "Yellow" groups are indicated in parenthesis.See Fig.4for sequences included in different Groups.